Signal peptide mutations in RANK prevent downstream activation of NF‐κB

Familial expansile osteolysis and related disorders are caused by heterozygous tandem duplication mutations in the signal peptide region of the gene encoding receptor activator of NF‐κB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF‐κB signaling in vitro, and it has been assumed that this accounts for the focal osteolytic lesions that are seen in vivo. We show here that constitutive activation of NF‐κB occurred in HEK293 cells overexpressing wild‐type or mutant RANK but not in stably transfected cell lines expressing low levels of each RANK gene. Importantly, only cells expressing wild‐type RANK demonstrated ligand‐dependent activation of NF‐κB. When overexpressed, mutant RANK did not localize to the plasma membrane but localized to extensive areas of organized smooth endoplasmic reticulum, whereas, as expected, wild‐type RANK was detected at the plasma membrane and in the Golgi apparatus. This intracellular accumulation of the mutant proteins is probably the result of lack of signal peptide cleavage because, using two in vitro translation systems, we demonstrate that the mutations in RANK prevent cleavage of the signal peptide. In conclusion, signal peptide mutations lead to accumulation of RANK in the endoplasmic reticulum and prevent direct activation by RANK ligand. These results strongly suggest that the increased osteoclast formation/activity caused by these mutations cannot be explained by studying the homozygous phenotype alone but requires further detailed investigation of the heterozygous expression of the mutant RANK proteins. © 2011 American Society for Bone and Mineral Research     

Processing of the NF‐κB2 precursor p100 to p52 is critical for RANKL‐induced osteoclast differentiation

Gene targeting of the p50 and p52 subunits of NF‐κB has shown that NF‐κB plays a critical role in osteoclast differentiation. However, the molecular mechanism by which NF‐κB regulates osteoclast differentiation is still unclear. To address this issue, we analyzed alymphoplasia (aly/aly) mice in which the processing of p100 to p52 does not occur owing to an inactive form of NF‐κB‐inducing kinase (NIK). Aly/aly mice showed a mild osteopetrosis with significantly reduced osteoclast numbers. RANKL‐induced osteoclastogenesis from bone marrow cells of aly/aly mice also was suppressed. RANKL still induced the degradation of IκBα and activated classical NF‐κB, whereas processing of p100 to p52 was abolished by the aly/aly mutation. Moreover, RANKL‐induced expression of NFATc1 was impaired in aly/aly bone marrow. Overexpression of constitutively active IKKα or p52 restored osteoclastogenesis in aly/aly cells. Finally, transfection of either wild‐type p100, p100ΔGRR that cannot be processed to p52, or p52 into NF‐κB2‐deficient cells followed by RANKL treatment revealed a strong correlation between the number of osteoclasts induced by RANKL and the ratio of p52 to p100 expression. Our data provide a new finding for a previously unappreciated role for NF‐κB in osteoclast differentiation. © 2010 American Society for Bone and Mineral Research     

Remifentanil post‐conditioning attenuates cardiac ischemia–reperfusion injury via κ or δ opioid receptor activation

Background: Ischemic pre‐ or post‐conditioning of the heart has been shown to involve opioid receptors. Remifentanil, an ultra‐short‐acting selective μ opioid receptor agonist in clinical use, pre‐conditions the rat heart against ischemia–reperfusion injury. This study investigates whether remifentanil post‐conditioning is also cardioprotective. Methods: Remifentanil post‐conditioning (5‐min infusion at 1, 5, 10 or 20 μg/kg/min) or ischemic post‐conditioning (three cycles of a 10 s reperfusion interspersed with a 10 s ischemia) was induced in an open‐chest rat heart model of ischemia and reperfusion injury, in the presence or absence of nor‐binaltorphimine, naltrindole or CTOP, specific κ, δ and μ opioid receptor antagonists, respectively. The same sequence of experiments was repeated in the isolated heart model using the maximal protective dose of remifentanil from the dose–response studies. Results: Both ischemic and remifentanil post‐conditioning reduced the myocardial infarct size relative to the control group in both models. This cardioprotective effect for both post‐conditioning regimes was prevented by the prior administration of nor‐binaltorphimine and naltrindole but not CTOP. The sole administration of the antagonists had no effect on the size of myocardial infarction. Conclusions: These results indicate that remifentanil post‐conditioning protects the heart from ischemia–reperfusion injury to a similar extent as of ischemic post‐conditioning. This protection involves κ and δ but not μ opioid receptor activation. This drug has great potential as a clinical post‐conditioning modality as it can be given in large doses without prolonged opioid‐related side effects.     

Activation of β‐Catenin Signaling in Articular Chondrocytes Leads to Osteoarthritis‐Like Phenotype in Adult β‐Catenin Conditional Activation Mice

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/β‐catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for β‐catenin in this disease has not been reported. Because tissue‐specific activation of the β‐catenin gene (targeted by Col2a1‐Cre) is embryonic lethal, we specifically activated the β‐catenin gene in articular chondrocytes in adult mice by generating β‐catenin conditional activation (cAct) mice through breeding of β‐catenin^{fx(Ex3)/fx(Ex3)} mice with Col2a1‐CreER^T2 transgenic mice. Deletion of exon 3 of the β‐catenin gene results in the production of a stabilized fusion β‐catenin protein that is resistant to phosphorylation by GSK‐3β. In this study, tamoxifen was administered to the 3‐ and 6‐mo‐old Col2a1‐CreER^T2;β‐catenin^fx(Ex3)/wt mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of β‐catenin protein was detected by immunostaining in articular cartilage tissues of β‐catenin cAct mice. In 5‐mo‐old β‐catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8‐mo‐old β‐catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in β‐catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp‐9, Mmp‐13, Alp, Oc, and colX, was significantly increased (3‐ to 6‐fold) in articular chondrocytes derived from β‐catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6‐fold increase) in these cells. In addition, we also observed overexpression of β‐catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of β‐catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA‐like phenotype. This study provides direct and definitive evidence about the role of β‐catenin in the development of OA.     

Endogenous TGF‐β activity limits TSLP expression in the intervertebral disc tissue by suppressing NF‐κB activation

Thymic stromal lymphopoietin (TSLP), an IL‐7‐like cytokine, is highly expressed in herniated disc (HD) tissue and may act as a key molecule for the initiation of macrophage recruitment into the tissue and natural resorption of HD. However, it remains unclear how TSLP expression is regulated in the intervertebral discs. This study showed that expression of TSLP and phosphorylated NF‐κB in HD tissue samples was inversely correlated with expression of phosphorylated Smad2/3 (an indicator of active TGF‐β signaling) and vice versa in posterior lumbar spinal fusion samples. The pharmacological blockades of endogenous TGF‐β activity induced TSLP expression in mouse intervertebral disc tissue culture, which was inhibited by NF‐κB inhibitors. Additionally, phosphorylation of Smad2/3 was constitutively detected in mouse intervertebral disc tissue in the steady states. Collectively, these results suggest that endogenous TGF‐β activity limits TSLP expression in intervertebral disc tissue in the steady states by suppressing NF‐κB activation. The findings reveal a regulatory mechanism how TSLP expression is induced in the intervertebral disc tissue and suggest a novel role of TGF‐β in maintaining the homeostasis of intervertebral disc tissue. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1144–1149, 2013     

Activation of mTORC1 in B Lymphocytes Promotes Osteoclast Formation via Regulation of β‐Catenin and RANKL/OPG

The cytokine receptor activator of nuclear factor‐κB ligand (RANKL) induces osteoclast formation from monocyte/macrophage lineage cells. However, the mechanisms by which RANKL expression is controlled in cells that support osteoclast differentiation are still unclear. We show that deletion of TSC1 (tuberous sclerosis complex 1) in murine B cells causes constitutive activation of mechanistic target of rapamycin complex 1 (mTORC1) and stimulates RANKL but represses osteoprotegerin (OPG) expression and subsequently promotes osteoclast formation and causes osteoporosis in mice. Furthermore, the regulation of RANKL/OPG and stimulation of osteoclastogenesis by mTORC1 was confirmed in a variety of RANKL‐expressing cells and in vivo. Mechanistically, mTORC1 controls RANKL/OPG expression through negative feedback inactivation of Akt, destabilization of β‐catenin mRNA, and downregulation of β‐catenin. Our findings demonstrate that mTORC1 activation‐stimulated RANKL expression in B cells is sufficient to induce bone loss and osteoporosis. The study also established a link between mTORC1 and the RANKL/OPG axis via negative regulation of β‐catenin. © 2016 American Society for Bone and Mineral Research.     

Advanced glycation end‐products induced VEGF production and inflammatory responses in human synoviocytes via RAGE‐NF‐κB pathway activation

Aging and diabetes are known to be the major cause to affect the progression of osteoarthritis (OA). Advanced glycation end products (AGEs) have been observed to accumulate in various organs especially in joint tissue and do damage to the joint tissue during aging and diabetes. Synovial angiogenesis and inflammation are observed across the full range of OA severity. The signaling pathway of AGEs on vascular endothelial growth factor (VEGF) production and inflammatory responses in synoviocytes are still unclear. Here, we investigated the role of receptor for AGEs (RAGE) and the signaling pathway involved in AGEs‐induced VEGF production and inflammatory responses in human synoviocytes. Human synoviocytes were cultured and treated with AGEs (25–100 µg/ml). AGEs significantly induced the protein expressions of cyclooxygenase‐2 (COX‐2) and VEGF and the productions of prostaglandin‐E2 (PGE2), VEGF, interleukin‐6 (IL‐6), and metalloproteinase‐13 (MMP‐13) in human synoviocytes in a dose‐dependent manner. Moreover, AGEs markedly activated the phosphorylations of IκB kinase (IKK)α/β, IκBα, and nuclear factor (NF)‐κB‐p65 proteins in human synoviocytes in a time‐dependent manner. Treatment with neutralizing antibody for RAGE statistically significantly decreased the AGEs‐induced increase in COX‐2, VEGF, PGE2, IL‐6, and MMP13 and AGEs‐activated NF‐κB pathway activation. Taken together, these findings indicate that AGEs are capable of inducing VEGF production and inflammatory responses via RAGE‐NF‐κB pathway activation in human synoviocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:791–800, 2016.